CONTRIBUTIONS AT THE PHARMACOGNOSTIC STUDY OF ELAEAGNUS ANGUSTIFOLIA L. SPECIES. NOTE I: POLIPHENOLIC COMPOUNDS STUDY OF FRUITS

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1 Contribuţii Botanice, XL, 2005 Grădina Botanică Alexandru Borza Cluj-Napoca CONTRIBUTIONS AT THE PHARMACOGNOSTIC STUDY OF ELAEAGNUS ANGUSTIFOLIA L. SPECIES. NOTE I: POLIPHENOLIC COMPOUNDS STUDY OF FRUITS Laura BUCUR 1, Constanţa SAVA 1, Viorica ISTUDOR 2 1 Universitatea Ovidius, Facultatea de Medicină Dentară şi Farmacie, Catedra de Farmacie, str. Ilarie Voronca, nr. 7, RO Constanta 2 Universitatea de Medicină şi Farmacie Carol Davila, Facultatea de Farmacie, str. Traian Vuia, nr. 7, RO Bucureşti Abstract: Using like vitamins (A, E, C) and proteins sources, the Elaeagnus angustifolia L. fruits have a reach composition [1]. After a preliminary chemical study was identified the poliphenolcarboxilic acid (ODP) and flavonoides (flavonosides, proantocianosides, tannins), moreover saccharides and mucilage. The quantitative analysis of ODP and certain flavonoides (flavonosides) was made using the spectrophotometric methods with a Turner apparatus beside caffeic acid and rutoside, like reference substances. The quantitative analysis show a medium contents in flavonoides as 0,13% couched in rutoside and ODP as 0,06% couched in caffeic acid. The antioxidant properties of ODP and flavonoides, known by reference materials [5], make to affirm that the drug could be interesting for capitalization in phytotherapie. Introduction Elaeagnus angustifolia L., Russian olive (Elaeagnaceae), is a specific plant in sandy soils area, spread in Dobrogea, on the sea cost area, but also in other region of the country [6]. The name of genus come by the Greek élaia who mean olive, because the fruit look like an olive and the name of specie suggest the slim aspect of the leaves. The fruit covered by the modified cell compose a false drupe. The external tissue is floury and the internal tissue is woody. There have an oval train, roundabout 1 cm length yellowish colour covered by silver hairs, with a floury and sweet pulp. The seed is covered by a hard endocarp with 8 black stripes alternated with 8 brown stripes [6]. While participate of the same family with Hippophäe rhamnoides L., sea buckthorn, witch fruits are considered the Romanian ginseng, the Russian olive is not used in phytotherapie through the floury aspect of the pulp and the presence of scaly hairs. Anyway is considered a useful plant because it fixes the ecosystems in the sea cost area and the fruits are very appreciated by different species of birds, especial in the cold season. The presence in a fruits of poliphenolic compounds (flavonozides and ODP), notified in reference materials [1] and the results of the preliminary research [3], establish us to deepen their study. The research objectives consist of: verification the identity of studied drug; confirmation of the presence to the poliphenolic compounds (flavonosides and ODP) in the native drug; the establishment of the quality of the drug by determination the contents of solubles substances in ethanol, flavonozides and ODP.

2 248 L. BUCUR, C. SAVA, V. ISTUDOR Material and Methods The drug is represented by the pulp of the fruits, reaping sunny period, in September 2004, from Constanta and environment. Separation the pulp to the hard kernel made manually, by scrap between fingers. The method using is pharmacognostic analysis. Verification of the fruits identity was made by macroscopic examination and on powder microscopic preparation, clarified with cloralhidrat solution 80%. The hystochemical analysis was made on the powder microscopic preparation in Steimetz reagents. The confirmation to the presence of flavonosides and ODP was made using specific identity reactions [4] with solution 5% obtaining by break-down the drug at the boiling with ethanol (P 1 ) and ethanol 50% (P 2 ). The preliminary quantitative analysis (dried evaporation, soluble substances in ethanol) was made according FR X [7]. The quantitative analysis for flavonosides and ODP was realized by spectrophotometric method beside the etalon curves of rutoside and caffeic acid. For flavonosides the method bases to obtaining the yellow internal complex with AlCl 3, who increases the yellowish colour of the solution. For ODP, the method bases to the phenol properties to form with nitros acid the nitroso derived who izomerises to oxime. This last one has a low acid character and dissolve in alkaline solutions, with appearance a red colour [4]. The determinations was made with Turner apparatus using solution 5%. The reproducibility of this method was verified by statistic calculation (r = 0,995). Results and Discussions The fruits situated in raceme to the young branches (fig. 1 a), have a fluffy aspect, oval form, white-yellow colour, about 1 cm length, 0,7-0,8 cm diameter and floury and sweet pulp (Fig. 1b). Fig. 1a: Young branches with fruits Fig. 1b: Fruits (detail) The microscopic examination show the big mezocarp cells (Fig. 2a) and hairs like a fan from epicarp. (Fig. 2b). The hystochemical examination confirms the accumulation at the base of the hairs of the liposolubles compounds (probably resinic acids), coloured in red (Fig. 3). The poliphenols quantitative analysis confirm the presence to flavonoides and ODP. The results obtaining are showed in Table 1.

3 PHARMACOGNOSTIC STUDY OF ELAEAGNUS ANGUSTIFOLIA, NOTE I 249 Fig. 2a: Mezocarp cells Fig. 2b: Hair like a fan Fig. 3: Accumulation area of liposolubles compounds Table 1: The identification of flavonosides and ODP CRT. REAGENTS PRODUCTS INTENSITY COMPOUNDS NR. 1. Arnow Red colour + ODP 2. HCl + Mg Orage-red colour + 3. AlCl 3 Increase the yellowish + colour Flavonosides 4. NaOH Increase the yellowish ++ colour 5. Oxalic acid/ boric UV fluorescence + acid The preliminary determinations show a dried evaporation between % who corresponding to the stipulations of FR X (max. 12%) and a considerable contents in soluble substances in ethanol ( ). The quantitative analysis for flavonoides show a contents about % couched in rutoside, calculated according to formula: m rutoside x 10-3 % flavonosides (g/g) = x 100 m pv were: m rutoside = weight of rutoside m pv = weight of drug in samples solutions (5.0234g).

4 250 L. BUCUR, C. SAVA, V. ISTUDOR The results are presented in figure 4 and table 2. Fig. 4: Standard curve for flavonosides quantitative analysis (reference substance = rutoside) and graphic representation of a samples Table 2: Results of flavonosides quantitative analysis V i V f A C f C i m rutoside Sample sample sample µg/ml C i =C f *V f /V i C i *100*10-3 ml ml 430 nm ppm µg/ml (ppm) = C i *0,1 mg/100 ml P P P P P Samples mean P 2, P 3, P % flavone g/g % The quantitative analysis for ODP show a contents about % couched in caffeic acid, calculated according to formula: % ODP (g/g) = were: m caffeic acid = weight of caffeic acid m pv = weight of drug in samples solutions (5g). The results are presented in figure 5 and table 3. m caffeic acid x 10-3 m pv x 100

5 PHARMACOGNOSTIC STUDY OF ELAEAGNUS ANGUSTIFOLIA, NOTE I 251 Fig. 5: Standard curve for ODP quantitative analysis (reference substance = caffeic acid) and graphic representation of a samples Table 3: Results of ODP determination V i V f A C f C i m caffeic acid Sample sample sample µg/ml C i =C f *V f /V i C i *100*10-3 ml ml 510 nm ppm µg/ml (ppm) = C i *0,1 mg/100 ml P P P P P P Samples mean P 2, P 4, P % ODP g/g % Conclusions The quantities of flavonosides (0.13%) and ODP (0.06%) are low but we could say that Elaeagni fructus would be capitalized in phytotherapie for antioxidants properties. REFERENCES 1. Bekker, N.P, Glushenkova, A. I. 2001, Components of certain species of the Elaeagnaceae family, Chemistry of natural compounds, 37, (2): Brad, I., 1995, Cătina albă (Hippophae rhamnoides) Gin-seng românesc. Poate fi utilizată şi antihiv- SIDA?, Acta Phytoterapica Romanica, II, (2): Bucur, L., Arcuş, M., Popescu, A., Vameşu,,S., Istudor, V., 2003, Pharmacobotanical characterisation of Elaeagnus angustifolia L. (Elaeagnaceae), Ovidius University Annals of Medical Science Pharmacy, I, (2), Ovidius University Press, Constanţa: Ciulei, I., Istudor, V., Palade, M., Niculete, E., Gârd, C., 1995, Analiza farmacognostică şi fitochimică a produselor vegetale, I, Ed. Tehnoplast Company SRL, Bucureşti: 78-82, Istudor, V., 1998, Farmacognozie. Fitochimie. Fitoterapie, Ed. Medicală, Bucureşti: 113, 155.

6 252 L. BUCUR, C. SAVA, V. ISTUDOR 6. Săvulescu, T. şi colaboratori, 1961, Flora R.P.R., IV, Ed Acad R.P.R., Bucureşti: *** Farmacopeea Română, 1993, Ed Medicală, Bucureşti: 1016, CONTRIBUŢII LA STUDIUL FARMACOGNOSTIC AL SPECIEI ELAEAGNUS ANGUSTIFOLIA L. NOTA 1 STUDIUL DERIVAŢILOR POLIFENOLICI DIN FRUCTE (Rezumat) Utilizate ca sursă de vitamine (A, E şi C) şi de proteine, fructele speciei Elaeagnus angustifolia L., ce reprezintă hrana păsărilor până iarna târziu, s-a presupus că ar prezenta şi un bogat conţinut în alte principii active. În urma unui studiu chimic preliminar s-au evidenţiat acizi polifenol-carboxilici (ODP-uri) şi flavonoide (flavonozide, proantocianozide, cateholi). Determinarea cantitativă a ODP-urilor şi a unora dintre flavonoide (flavonozide), s-a efectuat prin metode spectrofotometrice, la un aparat TURNER, faţă de acidul cafeic, respectiv rutozidă, ca substanţe de referinţă. În urma acestora s-a stabilit un conţinut mediu de 0,13% flavone exprimate în rutozidă şi de 0,06% ODPuri exprimate în acid cafeic. Proprietăţile antioxidante ale acizilor polifenol-carboxilici şi flavonelor, cunoscute din literatura de specialitate, ne determină să afirmăm că produsul ar putea prezenta interes pentru a fi valorificat în fitoterapie.